Pancreatic cancer is a devastating disease in need of novel therapeutics. To this end, the molecular hallmark of pancreatic cancer is an activating mutation in the small GTPase KRAS. Unfortunately, it has been difficult to pharmacologically inhibit KRas, prompting us to identify druggable proteins required for KRas oncogenesis. To this end, we discovered eNOS, one of three proteins in the Nitric Oxide Synthase family (composed of eNOS, nNOS and iNOS) that generate nitric oxide (NO), mediates oncogenic KRAS tumorigenesis. Specifically, eNOS is activated by oncogenic Ras, and moreover, knockdown of eNOS in human pancreatic cancer cell lines, or genetic ablation of the eNOS gene in mice, greatly retarded oncogenic Ras-driven tumor growth. Most importantly, small molecular weight inhibitors of NOS enzymes have been developed and tested in human clinical trials of septic and cardiogenic shock. Capitalizing on the development of NOS inhibitors for the treatment of other diseases, we tested and found that one such general NOS inhibitor impeded tumor growth of human pancreatic cancer cell lines and extended survival in the most aggressive mouse model of pancreatic cancer. Thus, eNOS is a bona fide target for pancreatic cancer that is druggable. There are no eNOS-specific small molecular weight inhibitors. While general NOS inhibitors are attractive molecules to explore, at least initially since they have already been tested in humans, they nevertheless also inhibit iNOS and nNOS. Two arguments suggest the importance of developing eNOS-specific inhibitors. First, eNOS is the NOS family member involved in pancreatic cancer. Second, as knockout of progressively more NOS family members results in more severe phenotypes, general NOS inhibitors may have undesirable off-target effects compared to inhibitors that specifically target eNOS. Given this, we propose to screen for eNOS inhibitors. We have developed a cell-based assay suitable for high throughput analysis that differentiates between cellular eNOS, iNOS and nNOS enzyme activity. We now propose to utilize this cell-based assay to screen a library of small molecules for their ability to inhibit cellular eNOS preferentially over nNOS and iNOS. Completion of these studies will provide novel eNOS inhibitors as a first step to specifically target this enzyme in pancreatic cancers. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a devastating disease in need of novel therapeutics. In this regard, our efforts to identify small molecular weight inhibitors of eNOS, an enzyme that is required for pancreatic tumorigenesis, provides a new avenue to treat this fatal disease.